This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Feiner, J. R. Articles by Schuyler, J. A. Search for Related Content PubMed PubMed Citation Articles by Feiner, J. R. Articles by Schuyler, J. A. Related Collections Resuscitation Neuroanesthesia Pharmacology

---

NEUROSURGICAL ANESTHESIA

Mild Hypothermia, but Not Propofol, Is Neuroprotective in Organotypic Hippocampal Cultures

Department of Anesthesia and Perioperative Care, University of California, San Francisco.

Address correspondence and reprint requests to Philip E. Bickler, MD, PhD, Sciences 255, Box 0542, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143–0542. Address e-mail to bicklerp{at}anesthesia.ucsf.edu

	Abstract

Top Abstract Introduction Methods Results Discussion Conclusions References

 
The neuroprotective potency of anesthetics such as propofol compared to mild hypothermia remains undefined. Therefore, we determined whether propofol at two clinically relevant concentrations is as effective as mild hypothermia in preventing delayed neuron death in hippocampal slice cultures (HSC). Survival of neurons was assessed 2 and 3 days after 1 h oxygen and glucose deprivation (OGD) either at 37°C (with or without 10 or 100 µM propofol) or at an average temperature of 35°C during OGD (mild hypothermia). Cell death in CA1, CA3, and dentate neurons in each slice was measured with propidium iodide fluorescence. Mild hypothermia eliminated death in CA1, CA3, and dentate neurons but propofol protected dentate neurons only at a concentration of 10 µM; the more ischemia vulnerable CA1 and CA3 neurons were not protected by either 10 µM or 100 µM propofol. In slice cultures, the toxicity of 100 µM N-methyl-D-aspartate (NMDA), 500 µM glutamate, and 20 µM -amino-5-methyl-4-isoxazole propionic acid (AMPA) was not reduced by 100 µM propofol. Because propofol neuroprotection may involve gamma-aminobutyric acid (GABA)-mediated indirect inhibition of glutamate receptors (GluRs), the effects of propofol on GluR activity (calcium influx induced by GluR agonists) were studied in CA1 neurons in HSC, in isolated CA1 neurons, and in cortical brain slices. Propofol (100 and 200 µM, approximate burst suppression concentrations) decreased glutamate-mediated [Ca²⁺]_i increases ([Ca²⁺]_i) responses by 25%–35% in isolated CA1 neurons and reduced glutamate and NMDA [Ca²⁺]_i in acute and cultured hippocampal slices by 35%–50%. In both CA1 neurons and cortical slices, blocking GABA_A receptors with picrotoxin reduced the inhibition of GluRs substantially. We conclude that mild hypothermia, but not propofol, protects CA1 and CA3 neurons in hippocampal slice cultures subjected to oxygen and glucose deprivation. Propofol was not neuroprotective at concentrations that reduce glutamate and NMDA receptor responses in cortical and hippocampal neurons.

IMPLICATIONS: The neuroprotective qualities of propofol are controversial. In rat hippocampal slice cultures, mild hypothermia (35°C), but not propofol, was protective after oxygen and glucose deprivation. Failure of propofol neuroprotection in this model may be related to relatively modest inhibition of glutamate receptor responses and excitotoxicity.

	Introduction

Top Abstract Introduction Methods Results Discussion Conclusions References

 
The neuroprotective qualities of propofol remain controversial. Several studies have demonstrated neuroprotective effects of propofol in focal ischemia in intact animals (1–4). Propofol reduces cell injury or injury surrogates in cellular preparations (5–9). In contrast, propofol failed to reduce postoperative neurologic deficits after cardiac surgery (10) and has failed to protect cells in some in vivo (11) and in vitro models (9,12) of cerebral ischemia. Propofol has properties that might be neuroprotective, including free radical scavenging (8), augmentation of -aminobutyric acid (GABA) receptor currents, and inhibition of N-methyl-D-aspartate (NMDA)-type glutamate receptor currents (13–15).

The neuroprotective actions of propofol, if they exist, may involve interactions between GABA_A receptors and glutamate receptors. When activated by drugs such as propofol (16), GABA_A receptors hyperpolarize neurons and thus decrease the voltage-dependent current flow through NMDA receptors. The magnitude of this interaction has not been clearly defined. In addition, propofol inhibits NMDA receptors directly, although weakly. NMDA receptors expressed in Xenopus oocytes are slightly (30%) inhibited at clinical concentrations (35 µM) of propofol (14). Orser et al. (15) reported the IC₅₀ of propofol on NMDA responses in cultured mouse neurons to be 160 µM, a concentration that produces electroencephalogram (EEG) burst-suppression in humans.

In contrast to the controversy surrounding the putative neuroprotective effects of propofol, mild hypothermia has been shown to consistently reduce neuronal death in reversible focal and global ischemia in different animal species and models of cerebral ischemia and also in in vitro preparations (17). The neuroprotective potency of anesthetics compared to mild hypothermia remains unclear, as there have been few studies directly comparing the two.

The purpose of this study was to compare the neuroprotective effects of 10 and 100 µM propofol to those of mild hypothermia (35°C) in hippocampal slice culture, an in vitro model widely used to evaluate neuroprotective strategies. In addition, we investigated the effects of 1–200 µM propofol on glutamate receptor activity in hippocampal slice cultures, in isolated hippocampal CA1 neurons, and in cortical brain slices to determine if neuroprotection (or lack thereof) is related to inhibition of glutamate receptor responses and whether the mechanism of receptor inhibition involves GABA_A receptors.

	Methods

Top Abstract Introduction Methods Results Discussion Conclusions References

 
All studies were approved by the University of California, San Francisco (UCSF) Committee on Animal Research and conform to relevant National Institutes of Health (NIH) guidelines.

The neuroprotective effects of propofol were evaluated in hippocampal slice cultures, which enable relatively long-term assessment of the survival of several different neuronal populations. Cultures were prepared by standard methods (18,19) as modified by Sullivan et al. (20). Briefly, Sprague-Dawley rats (8–12 days old, Simonsen Laboratories, Gilroy, CA) were anesthetized with 1%–2% halothane and given an intraperitoneal injection of ketamine (10 mg/kg) and diazepam (0.2 mg/kg). The rats were decapitated and the hippocampi were removed and placed in 4°C Gey’s Balanced Salt Solution (GBSS, UCSF Cell Culture Facility). Next, the hippocampi were transversely sliced (400 µm thick) with a tissue slicer (Siskiyou Design Instruments, Grants Pass, OR) and stored in GBSS containing 0.038 mg/mL ketamine at 4°C for 1 h (21). The slices were then transferred onto 30-mm diameter membrane inserts (Millicell-CM, Millipore, Bedford, MA), and put into 6-well culture trays with 1.5 mL of slice culture medium per well. The slice culture medium consisted of 50% minimal essential medium (Eagles with Earle’s balanced salt solution, UCSF Cell Culture Facility), 25% Earle’s balanced salt solution (UCSF Cell Culture Facility), 25% heat inactivated horse serum (Hyclone Laboratories, South San Francisco, CA) with 6.5 mg/mL glucose and 5 mM KCl. Slices were kept in culture for 7–14 days before study.

Cortical and hippocampal brain slices were prepared from 14–20 day old Sprague-Dawley rats anesthetized with 2% halothane in oxygen. For cortical slices the following protocol was used: after decapitation, cortices were rapidly dissected, glued with cyanoacrylate to a holder, and immersed in 1°C –3°C artificial cerebrospinal fluid (aCSF, Earle’s balanced salts, composition in mM: NaCl 116, NaHCO₃ 25, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.9, NaH₂PO₄ 0.9, glucose 20, pH 7.40 bubbled with 5% CO₂/95% O₂). Cortical slices (300–350 µm thick) were then prepared with a vibrating tissue slicer. Hippocampal slices were prepared similarly except that the entire hippocampus was removed and slices (400 µm) were made with a Mac-Ilwain tissue chopper (Stoelting, Inc, Wood Dale, IL). To prepare slices for measurements of [Ca²⁺]_i, they were transferred to vials of aCSF containing 1 µM fura-2 acetoxymethyl ester (fura-2 AM, Molecular Probes, Eugene, OR). Slices were transferred into fresh fura-2 AM solution after 30–60 min. After 1 h, dye-loaded slices had fluorescence signals that were 5–10 times background fluorescence. However, to permit better recovery from slicing trauma, slices were not studied until 2 h after slicing. During this period, slices were maintained at room temperature (25°C) in oxygenated aCSF.

Isolated pyramidal neurons from freshly prepared hippocampal slices were prepared by cutting out the CA1 region and placing this portion of the slice in media containing 0.1% trypsin for 30 min. The slice fragments were then transferred to Ca²⁺/Mg²⁺-free dissociation buffer and gently triturated by 2–3 passes through a fire-polished Pasteur pipette. Dissociated cells 50 µL were then pipetted into 150 µL of standard media on Cell-Tak (Collaborative Biomedical Products, Bedford, MA) coated coverslips. Glutamate receptor activity was measured in these cells 2–3 h after isolation.

Change in free cytosolic calcium ([Ca²⁺]_i) produced by application of NMDA or glutamate was used as an index of functional receptor activity. Change in [Ca²⁺]_i was used for this purpose because calcium flux through glutamate receptors is an integral part of the function of these ion channels. Fura-loaded slices were alternately excited with 340 and 380 nm light, and emitted light intensity at 510 nm was recorded. Change in this ratio was used as an index of changes in [Ca²⁺]_i. This method produces an index of calcium change that is independent of dye loading and avoids assumptions regarding the dissociation constant of fura-2 in slices and about maximal and minimal fluorescence ratios when fura is Ca²⁺-free and Ca²⁺-saturated (22).

To measure [Ca²⁺]_i, slices were fixed with fine suture to a nylon mesh holder and mounted in a capped fluorometer cuvette containing 2 mL gassed aCSF and a stir bar. PO₂ in the cuvette remained >200 mm Hg during study. Slices maintained stable cytosolic calcium levels and adenosine triphosphate concentrations for >30 min. The cuvette was mounted in the thermostatically controlled sample chamber of a Hitachi F-2000 fluorometer. The temperature in the cuvette was measured with a fine gauge thermocouple wire.

Changes in fura-2 fluorescence signals were measured during application of 100 µM NMDA or 1.0 mM glutamate. Calcium fluxes were verified as coming predominately from receptor linked ion channels in preliminary experiments: 1 µM tetrodotoxin, 0.5 µM conotoxin (N-type calcium channel toxin), 0.1 µM -agatoxin GIVA (P/Q type voltage-gated calcium channel toxin), or nimodipine (L-type calcium channel antagonist) did not alter calcium influx during NMDA application by more than 5%. Further, reduction in [Ca²⁺] in the perfusate or inclusion of 10 µM MK-801 reduced glutamate-induced calcium changes by approximately 90%. These studies established that calcium influx mechanisms other than those attributable to the NMDA receptor conductance were not significant.

Measurement of glutamate receptor-mediated calcium changes in CA1 neurons in hippocampal slices and in cultured hippocampal neurons were made with an inverted microscope and photomultiplier tube system as previously described (23). A perfusion system (Automate, Inc., San Francisco, CA) was used to deliver solutions of aCSF, glutamate (1–2 mM with dimethyl sulfoxide [DMSO] 0.35%), or glutamate plus 200 µM propofol (also containing vehicle, 0.35% DMSO). To enhance the NMDA component of the glutamate response, a reduced concentration of Mg²⁺ (0.2 mM) and 1 µM glycine was included in the solutions.

In vitro ischemia was simulated by hypoxia combined with glucose-free media (OGD). Before hypoxia, the slices were washed three times with glucose-free Hank’s balanced salt solution (HBSS). The cultures were then placed into a 2-L airtight Billups-Rothenberg Modular Incubator chamber (Del Mar, CA) through which 95% N₂/5% CO₂ gas preheated to 37°C was passed at 5–10 L per minute. The temperature of the chamber was kept at 37°C by passing preheated gas through the chamber and by placing a heat lamp over the chamber. The temperature inside the chamber was monitored with a thermocouple thermometer. After 10 min of gas flow the chamber was sealed and placed in a 37°C incubator ("normothermic group"). The partial pressure of oxygen was approximately 0–0.2 mm Hg, measured with a Clark-type oxygen electrode. For studies involving propofol, slices were rinsed with glucose-free HBSS containing 0, 10, or 100 µM propofol in 0.1% (final dilution) DMSO; these drugs remained in contact with the cultures for the duration of the OGD. No intralipid was present in any of our studies. After the insult, the culture tray was removed from the chamber, the anoxic-glucose-free HBSS was aspirated from the wells, and standard (oxygenated) slice culture media was added.

Cell viability was assessed with propidium iodide (PI) fluorescence (Molecular Probes, Eugene, OR). PI, a highly polar fluorescent dye, penetrates damaged plasma membranes and binds to DNA. Before imaging, slice culture media containing 2.3 µM PI was added to the wells of the culture trays. After 15 min the slices were examined with a Nikon Diaphot 200 inverted microscope (Nikon Corporation, Tokyo, Japan) and fluorescent digital images were taken using a SPOT Jr. Digital Camera (Diagnostic Instruments Inc., Sterling Heights, MI). Excitation light wavelength was 490 nm and emission was 590 nm. The sensitivity of the camera and intensity of the excitation light was standardized from day to day. PI fluorescence was measured in the dentate gyrus, CA1, and CA3 regions of the hippocampal slices. Slices were discarded if they showed any visible PI fluorescence in these regions after 7–10 days in culture. Slices were imaged before OGD (signal assumed to represent 0% cell death) and after 2 and 3 days after OGD. In previous studies, we found that maximum post-OGD death consistently occurs at about day 2 or 3 and declines over the next 11 days (20). Serial measurements of PI fluorescence intensity were made in predefined areas (manually outlining CA1, CA3, and dentate separately) for each slice using NIH Image software (developed at the US National Institutes of Health, Bethesda, MD and available on the Internet at http://rsb.info.nih.gov/nih-image/). Thus, cell death was followed in the same regions of each slice after simulated ischemia. After the measurement of PI fluorescence on the third post-OGD day, all the neurons in the slice were killed to produce a fluorescence signal equal to 100% neuron death in the regions of interest by adding 100 µM potassium cyanide and 2 mM sodium iodoacetate to the cultures for at least 20 min. Twelve to 24 h later, final images of PI fluorescence (equated to 100% cell death) were acquired. Percentage of dead cells at 0, 2, and 3 days post-OGD were then calculated based on these values. A linear relationship exists between cell death and PI fluorescence intensity (19,21).

Because we found that propofol reduced glutamate receptor-mediated calcium changes in hippocampal neurons, we tested whether propofol reduced cell death caused by glutamate receptor agonists. For these studies we used 100 µM propofol plus one of the following compounds: 100 µM NMDA, 500 µM glutamate, or 20 µM -amino-5-methyl-4-isoxazole propionic acid (AMPA). These concentrations of NMDA, glutamate and AMPA were chosen because they cause a similar degree of cell death in CA1 neurons as does 1 h of OGD. Survival of CA1, CA3, and dentate neurons in slice cultures 48 h after 1 h exposure to these compounds in air/5% CO₂ at 37°C was then determined with PI fluorescence.

Preliminary examination showed that the data on percentage survival of neurons in the different regions of the slices were not normally distributed. Therefore, the Kruskal-Wallis test followed by the Mann-Whitney U-test (JMP; SAS Institute, Cary, NC) was used to compare different treatment groups. Student’s t-tests or analysis of variance were used to compare glutamate responses in isolated neurons or brain slices. Differences were considered significant at P < 0.05.

	Results

Top Abstract Introduction Methods Results Discussion Conclusions References

 
During a 3-day period after 1 h OGD at 37°C, death was most frequent in CA1 neurons (50%–70%), intermediate in CA3 neurons (30%–40%), and least in dentate neurons (10%–30%), as shown in Fig. 1. Mild hypothermia during the OGD period (35°C) reduced subsequent cell death in each of these regions in the slices (Fig. 1, right panels) (P < 0.001 compared with OGD in 37°C slice cultures). Hypothermia alone (no OGD) with or without propofol did not alter cell death in the cultures compared to 37°C control cultures (Table 1).

View larger version (33K): [in this window] [in a new window]   Figure 1. Percent death of CA1, CA3, and dentate neurons in hippocampal slice cultures 2 and 3 days after oxygen and glucose deprivation (OGD). Each data point shows a mean ± SE. The left three panels show the effects 10 µM propofol on cell death after OGD. The panels on the right compare the effects of mild hypothermia (35°C) and 100 µM propofol on cell death after OGD. In all groups, 0.1% dimethyl sulfoxide was present. Statistical significance (P < 0.05) is indicated by the following symbols: *increased cell death compared with control (no OGD); reduction in cell death by mild hypothermia compared with OGD; **reduction in cell death by propofol/OGD compared with OGD.

View larger version (59K): [in this window] [in a new window]   Figure 2. Examples of propidium iodide staining of cell bodies (dark spots staining nucleus of dead cells) in hippocampal slice cultures 3 days after oxygen glucose deprivation (OGD). The upper panel shows the typical distribution of cell death in CA1, CA3, and dentate neuron regions. The middle panel shows an example of a slice that had 100 µM propofol present during the 1-h OGD period and the lower panel shows an image of a slice that was mildly hypothermic (35°C) during the 1-h OGD period. The confines of CA1, CA3, and dentate were similar in all three slices shown.

In both CA3 and dentate neurons, and to a lesser extent in CA1 neurons, 100 µM propofol (without OGD) was associated with increased cell death compared with the 10 µM propofol group. Although the degree of cell death in the 100 µM propofol groups was similar to that seen in slices exposed only to DMSO (Table 1), we cannot exclude the possibility that 100 µM propofol has some minor degree of toxicity in CA3 and dentate neurons.

Because glutamate receptors play a key role in hypoxia/glucose deprivation-induced cell death in slice cultures (20,24), we next tested whether cell death mediated by activation of glutamate receptors is reduced by propofol. To do this, we applied glutamate, NMDA, or AMPA to slice cultures and evaluated resulting cell death. Figure 3 shows the effects of propofol on cell death in CA1, CA3, and dentate neurons in hippocampal slice cultures 2 days after a 1-h exposure to these excitotoxins. Propofol 100 µM did not reduce the toxicity of any of these three compounds.

View larger version (18K): [in this window] [in a new window]   Figure 3. Propofol does not attenuate NMDA, AMPA or glutamate toxicity in slice cultures. Cultures were exposed to 100 µM NMDA, 20 µM AMPA, or 500 µM glutamate for 1 h in an atmosphere of air/5% CO2. Each group contained equal amounts of the vehicle dimethyl sulfoxide (0.1%). Cell death was determined 48 h later by the propidium iodide fluorescence method.

View larger version (47K): [in this window] [in a new window]   Figure 4. Propofol reduces glutamate and NMDA-elicited increases in [Ca2+]I ([Ca2+]I) in freshly prepared (studied 2 h after isolation) and cultured rat hippocampal slices (studied 10 days after isolation). Responses are expressed relative to those in control slices. Error bars show ± 1 SE and the numbers of slices studied are indicated.

Responses to glutamate and NMDA were also evaluated in neurons isolated from hippocampal slices. Propofol (200 µM) decreased responses to application of glutamate in isolated hippocampal CA1 neurons by 90% ± 6% (n = 6, P < 0.001). An example of this decrease is shown in the upper panel of Figure 5, showing glutamate to an isolated CA1 neuron in the presence of propofol or after propofol washout. This experiment was done in a manner favoring Ca²⁺ influx through the NMDA receptor: 1 µM glycine and 0.2 mM Mg²⁺ were present in the perfusate containing 1 mM glutamate. To test whether the inhibition of glutamate responses depended on GABA_A receptors, picrotoxin was added to some of the test perfusates. Alone, picrotoxin had no effect on [Ca²⁺]_i, which was expected because no GABA is present to produce tonic GABA-receptor activation in this synapse-free preparation. Picrotoxin abolished propofol’s inhibitory effect on glutamate responses (n = 6, P < 0.001), as shown in the example in the bottom panel of Figure 5, suggesting a significant role for GABA_A receptors in the mechanism of propofol inhibition of glutamate receptor responses.

View larger version (21K): [in this window] [in a new window]   Figure 5. Examples of the effects of 200 µM propofol on glutamate-mediated increases in [Ca2+]i in isolated/cultured hippocampal CA1 neurons. These neurons were maintained in culture 24 h and did not synapse with other neurons. At the times indicated by the arrows, perfusate containing 1 mM glutamate, 1 µM glycine, 0.2 mM Mg2+, with or without propofol was applied for 10 s, followed by washout with standard perfusion medium, conditions favoring Ca2+ influx through the NMDA receptor. Panel A shows the inhibition of glutamate responses by propofol and Panel B shows that 100 µM picrotoxin blocks the propofol inhibition of glutamate responses.

View larger version (16K): [in this window] [in a new window]   Figure 6. The relationship between propofol concentration and percent inhibition of functional NMDA receptor activity in cortical slices. Functional NMDA receptor activity was measured by the increase in [Ca2+]i after application of 100 µM NMDA. Data points represent mean ± SE, with number of slices indicated above each point. Data were best fit with a logistic equation.

View larger version (41K): [in this window] [in a new window]   Figure 7. The GABA antagonist picrotoxin reduces propofol inhibition of glutamate receptor responses in cortical slices. Panel A shows responses to glutamate with or without 200 µM propofol or picrotoxin and Panel B shows the same with NMDA. Data show mean ± SE with number of slices indicated above the columns. Data are expressed relative to the responses of control slices exposed to glutamate or NMDA. *significant difference (P < 0.05) compared with control (analysis of variance).

	Discussion

Top Abstract Introduction Methods Results Discussion Conclusions References

 
We found that mild hypothermia, but not propofol, reduces delayed cell death in ischemia-vulnerable CA1 and CA3 neurons in hippocampal slice cultures. Mild hypothermia has consistently been found to be protective in a variety of intact animal models (17), as well as in in vitro models involving organotypic slice cultures (25). Therefore, the protective benefit of mild hypothermia was not unexpected. More remarkable was the minimal protective benefit of propofol at concentrations that depress the activity of glutamate receptors in hippocampal and cortical neurons.

Some, but not all, studies have found that propofol reduces ischemia-induced brain injuries. In some of the studies in which propofol was found to be protective there were unresolved questions concerning propofol’s neuroprotective efficacy. In studies with intact animals, propofol’s effects on ischemia-induced brain injuries have mainly been examined over short time periods after ischemia (1,2). Anesthetic neuroprotection may not be durable, and early assessment of infarction (even at 1 week) may underestimate the total injury at a later time (26). Thus, the protective effects of propofol may not have been examined for a long enough period after ischemia to confirm that it affords durable neuroprotection. A study with middle cerebral artery occlusion found no neuroprotection with propofol (11). A clinical trial of propofol-induced EEG burst suppression found no benefit in cognitive outcome after coronary artery bypass graft surgery (10).

With in vitro models of ischemia, propofol’s neuroprotective actions have mainly been assessed as an injury surrogates, such as failure of neurotransmission, rather than cell death. The results of our study are similar to previous in vitro studies with acutely made brain slices showing that propofol does not reduce acute neurotransmission injury in hippocampal slices at 37°C (9) or reduce excitoxicity from AMPA or NMDA (27). Amorim et al. (9) did report that propofol reduced hypoxia-induced neurotransmission damage at 39°C, a finding that was related to attenuation of hypoxia-induced changes in Ca²⁺, Na⁺ and K⁺.

Previous investigations have focused on the capacity of propofol to reduce the free-radical component of ischemic injury (5,6,8). Because hippocampal slice cultures may be relatively resistant to some forms of free-radical induced injury (28), our study may have reduced the chance of propofol to exert a neuroprotective effect based on attenuation of free-radical damage.

We cannot completely exclude a minor neurotoxic effect of 100 µM propofol on our slice cultures. As shown in Figure 1, slices exposed to this concentration of propofol showed 10%–15% cell death in CA3 and dentate after 72 hr. This effect was not seen in cultures exposed to 10 µM propofol or in cultures that were hypothermic during exposure to 100 µM propofol. However, 4%–14% cell death was observed in cultures only exposed to DMSO at 37°C (Table 1). Because of this, we believe that we are justified in concluding that neither 10 µM nor 100 µM propofol protects CA1 or CA3 neurons in slice cultures after OGD, although some toxicity may exist. It is likely that all anesthetics possess a dose-dependent combination of protective and toxic effects, which depend on model and duration of exposure.

Propofol reduced glutamate receptor responses in CA1 neurons (acutely isolated neurons, neurons in acutely prepared slices, and neurons in 10-day-old slice cultures) but it did not protect these cells after OGD. Glutamate receptors play an important role in the mechanism of ischemic neuron death both in intact animals and in in vitro models. Evidence suggests that anesthetic neuroprotection is based at least partly on inhibition of glutamate excitotoxicity. Volatile anesthetics such as isoflurane decrease NMDA receptor activity (29,30), increase glutamate uptake (31), and suppress glutamate release (32,33). Glutamate receptor antagonists protect against cerebral injury in focal (34–36) and transient global ischemia (37) in intact rodent models. However, propofol is a relatively weak antagonist of the NMDA receptor, both in comparison to antagonists such as MK-801 in reducing cell death from NMDA exposure in cultured neurons (38) and in terms of the percentage inhibition of NMDA receptor activity at clinical concentrations (14,15). In Figure 6, the EC₅₀ for propofol inhibition of NMDA responses in cortical slices is approximately 100 µM, somewhat less than the 160 µM concentration reported by Orser et al. (15). Propofol may thus lack the glutamate receptor antagonism potency required for neuroprotection by an antiexcitotoxicity mechanism. This conclusion is also consistent with the data presented in Figure 3 showing that 100 µM propofol does not reduce the neurotoxicity of AMPA, NMDA, or glutamate in slice cultures and with previous studies in acutely prepared hippocampal slices (27).

The GABA agonist properties of propofol (16) are another reason we expected propofol to exhibit neuroprotection in CA1 neurons. Most of propofol’s inhibition of glutamate receptor responses is mediated via GABA receptors because most of the antagonism of glutamate receptor responses by propofol is removed in the presence of picrotoxin (Figs. 3 and 5). Abundant evidence suggests that GABA receptor agonists are neuroprotective against ischemia-induced injuries in the hippocampus (39). This is true in our slice culture model because the GABA agonist muscimol is neuroprotective and isoflurane neuroprotection is partly dependent on GABA receptors (40). Because of propofol’s relatively weak antagonism of glutamate receptor responses, it is possible that clinical concentrations of propofol are insufficient to produce a neuroprotective effect based on GABA receptors in our slice culture model.

	Conclusions

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Although clinical concentrations of propofol decreased glutamate receptor activity in CA1 neurons in hippocampal slices, they did not reduce death of CA1 neurons after OGD or after exposure to glutamate receptor agonists. In contrast, mild hypothermia provided significant reduction in cell death. Moderate reduction of glutamate receptor responses may not be synonymous with neuroprotection in hippocampal slice models of cerebral ischemia.

	Acknowledgments

 
Supported, in part, by grants from the National Institutes of Health (RO1 GM 52212 to PEB) and the Genentech Foundation (to SE).

	References

Top Abstract Introduction Methods Results Discussion Conclusions References

 

1. Kochs E, Hoffman WE, Werner C, et al. The effects of propofol on brain electrical activity, neurologic outcome, and neuronal damage following incomplete ischemia in rats. Anesthesiology 1992; 76: 245–52.[Web of Science][Medline]
2. Young Y, Menon DK, Tisavipat N, et al. Propofol neuroprotection in a rat model of ischaemia reperfusion injury. Eur J Anaesthiol 1997; 14: 320–6.
3. Yano T, Nakayama R, Ushijima K. Intracerebroventricular propofol is neuroprotective against transient global ischemia in rats: extracellular glutamate level is not a major determinant. Brain Res 2000; 883: 69–76.[Web of Science][Medline]
4. Ito H, Watanabe Y, Isshiki A, Uchino H. Neuroprotective properties of propofol and midazolam, but not pentobarbital, on neuronal damage induced by forebrain ischemia, based on the GABAA receptors. Acta Anaesthesiol Scand 1999; 43: 153–62.[Web of Science][Medline]
5. Sagara Y, Hendler S, Khoh-Reiter S, et al. Propofol hemisuccinate protects neuronal cells from oxidative injury. J Neurochem 1999; 73: 2524–30.[Web of Science][Medline]
6. Daskalopoulos R, Korcok J, Farhangkhgoee P, et al. Propofol protection of sodium-hydrogen exchange activity sustains glutamate uptake during oxidative stress. Anesth Analg 2001; 93: 1199–204.[Abstract/Free Full Text]
7. Hans P, Bonhomme V, Collette J, et al. Propofol protects cultured rat hippocampal neurons against N-methyl-D-aspartate receptor-mediated glutamate toxicity. J Neurosurg Anesthesiol 1994; 6: 249–53.[Web of Science][Medline]
8. Grasshoff C, Gillessen T. The effect of propofol on increased superoxide concentration in cultured rat cerebrocortical neurons after stimulation of N-methyl-D-aspartate receptors. Anesth Analg 2002; 95: 920–2.[Abstract/Free Full Text]
9. Amorim P, Chambers G, Cottrell J, Kass IS. Propofol reduces neuronal transmission damage and attenuates the changes in calcium, potassium, and sodium during hyperthermic anoxia in the rat hippocampal slice. Anesthesiology 1995; 83: 1254–65.[Web of Science][Medline]
10. Roach GW, Newman MF, Murkin JM, et al. Ineffectiveness of burst suppression therapy in mitigating perioperative cerebrovascular dysfunction. Multicenter Study of Perioperative Ischemia (McSPI) Research Group. Anesthesiology 1999; 90: 1255–64.[Web of Science][Medline]
11. Tsai YC, Huang SJ, Lai YY, et al. Propofol does not reduce infarct volume in rats undergoing permanent middle cerebral artery occlusion. Acta Anaesthesiol Sin 1994; 32: 99–104.[Medline]
12. Zhan R, Qi S, Wu C, et al. Intravenous anesthetics differentially reduce neurotransmission damage caused by oxygen-glucose deprivation in rat hippocampal slices in correlation with N-methyl-D-aspartate receptor inhibition. Crit Care Med 2001; 29: 808–13.[Web of Science][Medline]
13. Wakasugi M, Hirota K, Roth SH, Ito Y. The effects of general anesthetics on excitatory neurotransmission in area CA1 of the rat hippocampus in vitro. Anesth Analg 1999; 88: 676–80.[Abstract/Free Full Text]
14. Yamakura T, Sakimura K, Shimoji K, Mishina M. Effect of propofol on various AMPA-, kainate- and NMDA-selective glutamate receptor channels expresses in Xenopus oocytes. Neurosci Lett 1995; 188: 187–90.[Web of Science][Medline]
15. Orser BA, Bertlik M, Wang LY, MacDonald JF. Inhibition by propofol (2,6 di-isopropylphenol) of the N-methyl-D-aspartate subtype of glutamate receptor in cultured hippocampal neurones. Br J Pharmacol 1995; 116: 1761–8.[Web of Science][Medline]
16. Orser BA, Wang L-Y, Pennefather PS, MacDonald JF. Propofol modulates activation and desensitization of GABA_A receptors in cultured murine hippocampal neurons. J Neurosci 1994; 14: 7747–60.[Abstract]
17. Kataoka K, Yanase H. Mild hypothermia: a revived countermeasure against ischemic neuronal damages. Neurosci Res 1998; 32: 103–17.[Web of Science][Medline]
18. Stoppini L, Buchs PA, Muller D. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 1991; 37: 173–82.[Web of Science][Medline]
19. Laake JH, Haug F-M, Weiloch T, Ottersen OP. A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence. Brain Res Brain Res Protoc 1999; 4: 173–84.[Medline]
20. Sullivan BS, Leu D, Taylor DM, et al. Isoflurane prevents delayed cell death in an organotypic slice culture model of cerebral ischemia. Anesthesiology 2002; 96: 189–95.[Web of Science][Medline]
21. Newell DW, Barth A, Papermaster V, Malouf AT. Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures. J Neuroscience 1995; 15: 7702–11.[Abstract]
22. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440–50.[Abstract/Free Full Text]
23. Bickler PE, Hansen BM. Hypoxia-tolerant neonatal CA1 neurons: relationship of survival to evoked glutamate release and glutamate receptor-mediated calcium changes in hippocampal slices. Dev Brain Res 1998; 106: 57–69.[Medline]
24. Newell DW, Malouf AT, Franck JE. Glutamate-mediated selective vulnerability to ischemia is present in organotypic cultures of hippocampus. Neurosci Lett 1990; 116: 325–30.[Web of Science][Medline]
25. Adamchik Y, Frantseva MV, Weisspapir M, et al. Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures. Brain Res Brain Res Protoc 2000; 5: 153–8.[Medline]
26. Kawaguchi M, Kimbro JR, Drummond JC, et al. Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischemia. Anesthesiology 2000; 92: 1335–42.[Web of Science][Medline]
27. Zhu H, Cottrell JE, Kass IS. The effect of thiopental and propofol on NMDA- and AMPA-mediated glutamate excitotoxicity. Anesthesiology 1997; 87: 944–51.[Web of Science][Medline]
28. Keynes RG, Duport S, Garthwaite J. Hippocampal neurons in organotypic slice culture are highly resistant to damage by endogenous and exogenous nitric oxide. Eur J Neurosci 2004; 19: 1163–73.[Web of Science][Medline]
29. Bickler PE, Buck LT, Hansen BM. Effects of isoflurane and hypothermia on glutamate receptor-mediated calcium influx in brain slices. Anesthesiology 1994; 81: 1461–9.[Web of Science][Medline]
30. Yang J, Zorumski CF. Effects of isoflurane in N-methyl-D-aspartate gated ion channels in cultured rat hippocampal neurons. Ann N Y Acad Sci 1991; 625: 287–9.[Web of Science][Medline]
31. Miyazaki H, Nakamura Y, Arai T, Kataoka K. Increase of glutamate uptake in astrocytes: a possible mechanism of action of volatile anesthetics. Anesthesiology 1997; 86: 1359–66.[Web of Science][Medline]
32. Patel PM, Drummond JC, Cole DJ, Goskowicz RL. Isoflurane reduces ischemia-induced glutamate release in rats subjected to forebrain ischemia. Anesthesiology 1995; 82: 996–1003.[Web of Science][Medline]
33. Bickler PE, Buck L, Feiner JR. Volatile and intravenous anesthetics decrease glutamate release from cortical brain slices during anoxia. Anesthesiology 1995; 83: 1233–40.[Web of Science][Medline]
34. Sarraf-Yazdi S, Sheng H, Miura Y, et al. Relative neuroprotective effects of dizocilpine and isoflurane during focal ischemia in the rat. Anesth Analg 1998; 87: 72–8.[Abstract/Free Full Text]
35. Park C, Nehls D, Graham D, et al. Focal cerebral ischemia in the cat: treatment with the glutamate antagonist MK-801 after the induction of ischemia. J Cereb Blood Flow Metab 1988; 8: 757–62.[Web of Science][Medline]
36. Dezsi L, Greenberg J, Hamar J, et al. Acute improvement in histological outcome by MK-801 following cerebral ischemia and reperfusion in the cat independent of blood flow changes. J Cereb Blood Flow Metab 1992; 12: 390–9.[Web of Science][Medline]
37. Gill R, Foster AC, Woodruff GN. MK-801 is neuroprotective in gerbils when administered during the post-ischemic period. Neuroscience 1988; 25: 847–55.[Web of Science][Medline]
38. Kudo M, Aono M, Lee Y et al. Effects of volatile anesthetics on N-methyl-D-aspartate excitotoxicity in primary rat neuronal-glial cultures. Anesthesiology 2001; 95: 756–65.[Web of Science][Medline]
39. Schwartz-Bloom RD, Miller KA, Evenson DA, et al. Benzodiazepines protect hippocampal neurons from degeneration after transient cerebral ischemia: an ultrastructural study. Neuroscience 2000; 98: 471–84.[Web of Science][Medline]
40. Bickler PE, Warner DS, Stratmann G, Schuyler J. GABA receptors contribute to isoflurane neuroprotection in organotypic hippocampal cultures. Anesth Analg 2003; 97: 564–71.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Sigaut, V. Jannier, D. Rouelle, P. Gressens, J. Mantz, and S. Dahmani The Preconditioning Effect of Sevoflurane on the Oxygen Glucose-Deprived Hippocampal Slice: The Role of Tyrosine Kinases and Duration of Ischemia Anesth. Analg., February 1, 2009; 108(2): 601 - 608. [Abstract] [Full Text] [PDF]

				H. Cimarosti and J. M. Henley Investigating the Mechanisms Underlying Neuronal Death in Ischemia Using In Vitro Oxygen-Glucose Deprivation: Potential Involvement of Protein SUMOylation Neuroscientist, December 1, 2008; 14(6): 626 - 636. [Abstract] [PDF]

				S. J. Howell Carotid endarterectomy Br. J. Anaesth., July 1, 2007; 99(1): 119 - 131. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Feiner, J. R. Articles by Schuyler, J. A. Search for Related Content PubMed PubMed Citation Articles by Feiner, J. R. Articles by Schuyler, J. A. Related Collections Resuscitation Neuroanesthesia Pharmacology